SUMMARY (See instructions): Allograft rejection, acute and chronic, is a common complication of organ transplantation, significantly Increases medical costs, but more Importantly It dramatically and negatively affects the lives of patients. In the previous grant period we have carried out a large-scale mass spectrometry-based proteomic analysis in clinical samples from patients undergoing acute and the chronic kidney transplant rejection and compared these to patients with healthy transplants. We have studied biopsies and peripheral blood Including purified, activated T and B cells. We have identified 1,000s of proteins differentially expressed among the patient cohorts with various clinical outcomes. Many of these proteins belong to key cellular, innate immunity, inflammatory and tissue remodeling pathways and are very likely the first proteomic signatures of rejection. We propose to continue our work with transplant proteomics and to extend our studies Into the areas of clinical proteomic assay development, heart transplant rejection, and human lymphocyte phosphoproteomlcs. In Specific Aim #1 we are proposing to develop a diagnostic, high-throughput clinical proteomic blood-based assay for acute and chronic rejection based on selected reaction monitoring (SRM) targeted mass spectrometry. The large-scale discovery efforts from the previous grant period yielded many potential targets for candidate biomarkers that will be validated via SRM and developed into a diagnostic assay. In Specific Aim #2 we are going to perform large-scale discovery-based mass spectrometry proteomic analysis of acute and chronic heart transplant rejection. We hope to unravel some of the molecular mechanisms and biological functions that are indicative of heart transplant rejection by further comparing our new data to the proteomic data from the kidney transplant system. Specific Aim #3 is geared toward the phosphoproteomic analysis of transplant rejection. We are proposing to Identify and quantify proteins that become phosphorylated as a response to transplant rejection. We will profile the CD4 T and B cells from patients undergoing transplant rejection and Investigate signaling changes taking place due to the transplant related Immunosuppression, T and B cell activation and Inflammatory processes.